Reduced glutathione (GSH) is one of the cell's major defenses against oxidative stress. Lowered GSH levels have been implicated in susceptibility to numerous complex diseases (including neurodegenerative disorders, cancer, diabetes mellitus, cataracts, and AIDS) plus toxicity to environmental chemicals and heavy metal ions such as cadmium. Levels of GSH vary 10-fold between different cell types. The rate-limiting step in GSH biosynthesis is glutamate-cysteine ligase (GCL). GCL activity exists as the GCL catalytic subunit (GCLC) or as the GCL holoenzyme, a heterodimer composed of GCLC and a modifier subunit GCLM. Using gene targeting, we have generated conventional Gclm(-/-) and Cre-inducible Gclc(-/-) knockout mouse lines. GSH levels in Gclm(-/-) mice are only approximately 10% of that in Gclm(+/+) littermates in all tissues surveyed, yet, surprisingly, they are viable and fertile. Liver-specific Gclc(-/-) mice die by age 4 weeks, but can be rescued by N-acetylcysteine in the drinking water. With these mice, we are in a unique position to address the hypothesis that Gclm(-/-) mice will be susceptible to both environmental and endogenous toxicants because although the amount of GCLC controls the potential maximum level of cellular GSH, the GCLC/GCLM ratio determines the actual level. Thus, we will: [a] Evaluate endogenous, genotoxicity and oxidative stress in untreated Gclm(+/+) and Gclm(-/-) mice; [b] Assess cadmium-induced liver and kidney toxicity in Gclm(+/+) and Gclm(-/-) mice; and [c] Dissect the role of GCLC and GCLM in controlling GSH levels through inducible expression of GCLC and GCLM in double-knockout Gclc/Gclm(-/-) immortalized hepatocytes. By way of these studies, we will define further the role of GCLM and GSH during both heavy metal-induced as well as endogenously-induced oxidative stress, while evaluating the Gclm(-/-) mouse as a model for a compromised oxidative stress response. Further, we will delineate the roles of GCLC and GCLM in controlling GSH levels. These studies will provide valuable insight into understanding the etiology, preventive medicine, and the possible development of therapeutic intervention in the above-mentioned diseases and toxicities. For example, studies suggest that human genetic differences exist in toxicity to cadmium and other environmental toxicants that cause oxidative stress; moreover, the GCLC and GCLM genes are highly polymorphic. Our proposed research should therefore help focus future genotype-phenotype association studies between the appropriate DNA variant sites in the GCLC and GCLM genes and disease.